Correspondence Address:Hassan A KhodairDepartment of Dermatology and Andrology, Assistant Professor of Clinical Pathology, Faculty of Medicine, Al-Azhar University Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/1110-6530.123932

Abstract

BackgroundStudies suggest that reactive oxygen species (ROS) attack the integrity of DNA in the sperm nucleus by causing base modifications, DNA strand breaks, and chromatin cross-linking. Sperm DNA damage analysis may reveal hidden sperm DNA abnormalities in infertile men with normal standard semen analysis values who were diagnosed with idiopathic infertility.Aim of the workWe explored the levels of ROS and their correlation with sperm DNA damage in patients with idiopathic male infertility.Patients and methodsA total of 93 men were included in this study. Among them, 68 presented to our Andrology outpatient clinic with idiopathic infertility and were selected, and 25 were healthy fertile men, who were assigned to the control group. Both groups were subjected to the following laboratory investigations: semen analysis including peroxidase test, measurement of ROS levels by chemiluminescence assay, and sperm DNA damage assessment by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).ResultsThere were no significant differences in the semen parameters between idiopathic infertile men and controls. However, ROS and sperm DNA damage levels were significantly higher in idiopathic infertile men compared with controls ( P ͳ 0.001). In addition, the results showed a strong positive correlation between ROS levels and the percentage of sperm DNA damage ( P ͳ 0.001).ConclusionTraditional semen analysis does not reveal seminal defects at the molecular level that might be induced by ROS. Therefore, our results suggest that standard semen analysis should be coupled with measurement of ROS and assessment of DNA integrity in cases of idiopathic male infertility as even sperms with normal morphology and motility may harbor DNA damage.

In the etiology of male infertility, there is growing evidence that damage to spermatozoa by reactive oxygen species (ROS) play a key role [1] . Spermatozoa contain large quantities of polyunsaturated fatty acids. Therefore, they are susceptible to ROS-induced membrane lipid peroxidation, which may result in impairment of the integrity and functioning of the sperm plasma membrane [2] . In addition, supraphysiological ROS levels are also known to cause significant DNA damage to both mitochondrial [3] and nuclear genomes of human spermatozoa [4] . In such instances, these reactive oxygen metabolites attack DNA bases (particularly guanine) and phosphodiester backbones, destabilizing this molecule and creating the cellular conditions that ultimately result in DNA fragmentation [5] . The principal sources of endogenous ROS in semen are leukocytes and abnormal spermatozoa [6] . A plausible hypothesis recently suggested that, in most cases, the sperm DNA is attacked mainly by mitochondrial ROS originating from functionally defective spermatozoa [7] . Spermatozoa have limited defense mechanisms against oxidative attack on their DNA mainly because of the complex packaging arrangement of DNA [8] . Sperm DNA integrity is necessary for accurate transmission of genetic information and birth of healthy offspring [9] . It is critical for normal embryogenesis, fetal well being, and success of assisted reproductive techniques [10] . High ROS levels are also known to cause pronuclear block and impair cleavage, leading to production of morphologically abnormal blastomeres [11] . The other causes of DNA damage and oxidative stress are exposure to xenobiotics and environmental pollutants, varicocele, abortive apoptosis, infection, inflammation, high testicular temperature, and exposure to electromagnetic radiation [12] . Previous studies have shown that ROS levels are elevated in about 68% of infertile men [13] , and this is one of the most important factors in the etiology of DNA damage [3] . In addition, even sperm with normal morphology and motility may harbor DNA damage [14] . A large number of apparently normal men have difficulty impregnating their partners even when their fertility status on routine semen analysis is considered normal. These cases are classified as idiopathic infertility and represent about 60-75% of male infertility [15] . Therefore, we have speculated that the presence of oxidative damage may be the cause behind idiopathic infertility in normozoospermic men.

Aim of the study

The aim of the present study was to investigate the levels of ROS in patients with idiopathic male infertility with normal semen parameters and their correlation with sperm DNA damage.

Patients and methods

This study was conducted during the period from June 2011 to May 2013 and included 93 men who had been attending the Andrology outpatient clinic at Damiatta University Hospital, Al-Azhar University. Among them 68 had idiopathic infertility for a duration of 2-7 years, with normal semen parameters, and 25 were healthy fertile men who had recently fathered children; they were assigned to control group. Both groups were comparable in terms of age, and none of the men in either group had significant health problems. All participants underwent complete medical history taking with special reference to their occupation - that is, potential factors capable of impairing spermatogenesis - and andrological examination was performed for all study participants. Participants currently on any medication or antioxidant supplementation, as well as smokers, were excluded. In addition, patients suffering from varicocele and acute infection or leukocytospermia were also excluded from the study because of their well-known high seminal ROS levels. The fertilizing capacity of the partners of the infertile men was assessed, by hormonal assays and folliculometry, and was found to be functionally normal. Both groups were subjected to the following laboratory investigations: Semen analysis, including the peroxidase test to exclude leukocytospermia, measurement of ROS levels by a chemiluminescence assay, and sperm DNA damage assessment by terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).

Laboratory evaluation

Semen collection and evaluation

All semen samples were produced by masturbation after 3-5 days of abstinence and were collected in a sterile plastic container and incubated at 37°C after collection. The following routine parameters were assessed in the liquefied semen samples, according to the methods described in the WHO laboratory manual [16] : Volume, pH, color, liquefaction status, agglutination, sperm concentration, percentage and grade of motility, sperm morphology, sperm vitality, total sperm count, and total sperm motility. Results were considered normal when the semen volume was greater than 2 ml, sperm density was greater than 20 × 10 6 /ml, there was more than 50% forward progressive motility, and there were less than 40% abnormal forms including dead sperm. Semen volume was measured in a calibrated test tube. Sperm count and motility were assessed using a hemocytometer and a light microscope. Motility was examined at ×400 magnification. At least 100 sperms were examined and motility was expressed as the proportion of sperms showing evidence of movement - that is, rapid progressive motility, slow progressive motility, or nonprogressive motility - using a scale from 1+ (slowest) to 4+ (fastest). To analyze sperm morphology, at least two slides were prepared from each fresh semen sample. Semen samples were smeared onto slides and air-dried; the semen cells were washed twice in PBS and applied onto microscope slides, which dried and fixed in 100% methanol; thereafter, they were stained using the Papanicolau staining technique. The morphology of 100-200 sperms/individual was assessed using a phase contrast microscope. The percentage of sperms with normal morphology, which was characterized by a normal head, a mid-piece, and a tail, was estimated. For the sperm vitality test, a drop of liquefied semen was mixed with two drops of 1% eosin Y solution. After 30 s, three drops of nigrosin solution (10%) was added to the mixture and mixed. A thin smear of the semen-eosin-nigrosin mixture was made within 30 s of adding nigrosin; the smear was allowed to air-dry and examined under a microscope thereafter (×400). One hundred spermatozoa were counted to express the percentage of live spermatozoa, which appear white.

Peroxidase test

Leukocytes were counted using the leucoscreen method. Briefly, one drop of semen was mixed with one drop of working solution (leucoscreen stain and hydrogen peroxide); the mixture was covered with a cover slip for 2 min, after which the results were read at a magnification of ×400. Peroxidase-positive cells were stained yellow to brown, whereas the other cells were stained pink [17] .

Measurement of reactive oxygen species

To 400 μl of liquefied neat semen, 10 μl of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione; Sigma, USA), prepared as 5 mmol/l stock in dimethyl sulfoxide, was added. A volume of 10ml luminol (5 mmol/l) in dimethyl sulfoxide served as blank. A mixture of H 2 O 2 (25 μl) and luminol (10 μl) was used as the positive control. All the samples were measured in duplicate and the average of the readings was taken. Levels of ROS were assessed by measuring luminol-dependant chemiluminescence on a single-detector luminometer (Sirius; Berthold Detection Systems GmbH, Pforzheim, Germany) in the integrated mode for 15 min [18] . The results were expressed as 10 4 counted photons per minute (cpm)/20 × 10 6 sperm. The cutoff value for an abnormal ROS level in a neat sample was greater than 0.2 × 10 4 cpm/20 × 10 6 .

Evaluation of sperm DNA fragmentation

DNA in spermatozoa was visualized by TUNEL, using the Cell Death Detection Kit with tetramethylrhodamine-labeled dUTP (Roche, Monza, Italy) according to the manufacturer's instructions. Ejaculated sperm samples were washed from seminal plasma by low-speed centrifugation (×200; 10 min), smeared onto microscope slides, air-dried, fixed with 4% paraformaldehyde in PBS at 4°C for 25 min, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. Fixed sperm smears were further processed for TUNEL as described. Spermatozoa with fragmented DNA were detected under an epifluorescence microscope with a ×100 oil-immersion objective. For quantitative evaluation, 500 spermatozoa in 50 randomly selected areas on three different microscope slides were evaluated for each sample, and the percentage of TUNEL-positive spermatozoa was determined. The cutoff value for the percentage of TUNEL-positive spermatozoa was 40% [19] .

General characteristics of both fertile and idiopathic infertile groups

The data showed no statistically significant differences in the mean age between both groups (P > 0.05). The mean age in the idiopathic infertile group and the fertile control group was 34.5 ± 3.5 years (range 26-46 years) and 33.5 ± 2.5 years (range 25-45 years), respectively, as shown in [Table 1].

The data showed no statistically significant differences in the semen parameters tested, most notably in seminal volume, pH, viscosity, liquefaction time, sperm concentration, motility, grade of motility, normal sperm morphology, sperm vitality, total sperm count, and total number of motile sperm in both groups. The results (mean ±SD; P > 0.05) are given in [Table 2].

The data showed no statistically significant differences in white blood cell (WBC) concentration between both groups. The mean number of WBCs (10 6 /ml) in the idiopathic infertile and the fertile control group was 0.05 ± 0.03 and 0.06 ± 0.02 106/ml (P > 0.05), respectively, as shown in [Table 3].

The results obtained in the present study show that the levels of ROS were significantly higher in neat semen of the idiopathic infertile group compared with the fertile control group. Among the idiopathic infertile men, 51 (75%) had elevated ROS levels (1.2 ± 0.04 × 10 4 compared with 0.11 ± 0.02 × 10 4 cpm/20 × 10 6 spermatozoa in the fertile control group; P ≤ 0.001). The results (mean ± SD) are given in [Table 4].

The results showed that 58 (85.3%) of idiopathic infertile group, had higher sperm DNA fragmentation in more than or equal to 80% of spermatozoa. DNA fragmentation were significantly higher in idiopathic infertile group compared with fertile control group, (42.9 ± 7.9 vs. 10.4 ± 3.8%), P ≤ 0.001). The results (mean ± SD) are given in [Table 5].

Traditional semen analysis does not reveal seminal defects at the molecular level that might be induced by ROS, which are associated with male infertility [19] . Our study found no statistically significant differences in semen parameters and WBC concentration (no leukocytospermia) between the idiopathic infertile group and the fertile control group (P > 0.05). However, our results showed that ROS levels were significantly higher among men with idiopathic infertility compared with fertile controls (P ͳ 0.001), which is in accordance with the findings of the study by Mayorga et al. [15] . In addition, our study found that sperm DNA fragmentation was significantly higher among men with idiopathic infertility compared with fertile controls (P ͳ 0.001), which is in accordance with the findings of the study by Fingerova et al. [20] . Our results show a strong positive correlation between ROS levels and sperm DNA fragmentation percentage (P ͳ 0.001), which is in accordance with the findings of the study by Mayorga et al. [15] . Our results support those of several studies that report impaired clinical outcomes on intracytoplasmic sperm injection (ICSI) with ejaculated spermatozoa in men with elevated nuclear DNA damage [21] and idiopathic recurrent pregnancy loss [22],[23] . Consequently, these findings provide an explanation on why patients with normal semen parameters can experience idiopathic infertility. Possibly, ROS levels may not be high enough to affect standard seminal parameters but may cause defects in other processes that are required for fertilization and are dependent on the functional integrity of both the sperm membrane and sperm DNA. Our study reports that normal semen analysis does not guarantee the fertilization potential of sperm, and studies have shown significant overlap in semen parameter values between fertile and infertile men [24] . Sperm production is only a part of the story and integrity of sperm chromatin and DNA is essential to ensure that the fertilizing sperm can support normal embryonic development of the zygote. This study highlights the need to assess all infertile men with normal or abnormal semen parameters for sperm DNA damage and ROS levels. This will help predict future pregnancy outcome or explain previous assisted reproduction technique (ART) failure or early idiopathic pregnancy losses.